DESCRIPTION: As methods for gene expression measurement have improved, there has been a growing appreciation that biological behavior in single cells can vary dramatically in ways not captured by conventional bulk assays. Single molecule RNA FISH has developed into a powerful and generally applicable method for single cell gene expression analysis, having been adopted by dozens of labs worldwide. However, while it is able to precisely locate individual RNA molecules in single cells, it has not been able to discriminate single base differences on those RNA, thus precluding many potential biological applications. Such applications include differences in expression between the maternal and paternal alleles of a gene, which typically only differ by single nucleotides. These expression differences are relevant in many areas of genetics, including gene imprinting, in which only the maternal or paternal copy of the gene expresses. Many of these issues arise in biological contexts (e.g., complex and heterogeneous tissues such as brain and placenta) that make even single cell biochemical assays problematic because they do not preserve the spatial context of the cells in question. Thus, these biological areas of research have been unable to harness many of the advances that single cell biology has conferred. Recently, we have developed a method that enables us to distinguish individual RNAs based on single base differences. The goal of this proposal is to quantitative validate this method, first in cell lines and then in mouse and human tissues and then applying to specific biological questions that underscore the utility of the approach. Methodologically, we perform a series of experiments aimed at understanding the parameters relevant to our method with the goal of making the method robust, reliable and easily accessible to other researchers. In aim 1, we will perform these experiments in cell lines to characterize the probes themselves and develop the capability to measure multiple single nucleotide variants simultaneously within single cells. In aim 2, we will apply our method to tissue sections from mouse and human with the goal of solving common problems specific to tissue, such as RNA degradation and high background. As an application demonstrating the utility of our method, we will study Beckwith-Wiedemann Syndrome, a genetic disorder in which loss of appropriate imprinting leads to a highly varied phenotype. We will study the manifestations of this disorder by examining allele-specific expression in developing mouse tissues and then in de-identified human patient samples, examining how cell-to-cell variability in imprinting leads to the disease phenotype.